ABSTRACT: The overarching goal is to improve outcomes in allogeneic stem cell transplantation by identifying and enriching donor dendritic cell (DC) populations that reduce graft-versus-host disease and improve survival. The current proposal is grounded on published findings that the content of donor DC in the allograft and in the blood of allo-transplant recipients have a significant impact on clinical outcomes. Preclinical data from murine BMT model systems demonstrate that donor plasmacytoid dendritic cells (pDC) from marrow but not G-CSF mobilized grafts protect against GvHD, home to the thymus, and limit GvHD in an CCR9-, IL12-, and IDO- dependent manner. The question of optimizing immune cells in the graft is highly significant, as patients transplanted with more marrow donor pDC have 20% better survival, with less chronic GvHD, and without increased risk of relapse compared with recipients of fewer donor pDC. Unanswered questions that limit application of these exciting observations to widespread clinical practice include resolving heterogeneity of DC in marrow and cytokine-mobilized grafts to identify the critical subset(s) that confer favorable transplant outcomes; defining the mechanism by which donor DC regulate GvHD; and identifying cost-effective procedures for mobilization and harvesting optimal hematopoietic progenitor grafts requiring minimal ex vivo manipulation. To address these questions, and make progress towards our overall goal, we propose three Specific Aims: Aim 1. To define the mechanisms and molecular profile of donor DCs that limit GvHD. Hypothesis: Murine and human bone marrow contain immunosuppressive DC subset(s) that are present at higher frequencies than in G-CSF-mobilized grafts, that inhibit T cell activation in response to allo-antigen, and that limit GvHD. Aim 2. To enhance the immuno-regulatory potency of DCs in allotransplantation. Hypothesis: Treatment of donors with a short-course of Flt3-L will increase the numbers and immunological potency of immature donor DC that limit GvHD following transplantation to allogeneic recipients. Aim 3. To determine how homing and persistence of donor-derived DC affects GvHD and GvL. Hypothesis: donor pDC that home to hemato-lymphoid tissue in transplant recipients support tolerance via negative selection of allo-reactive T cells in the thymus and/or the generation of Treg. The proposed research will use single cell RNAseq and mass cytometry to define clusters of DC from mouse and human marrow and cytokine-mobilized grafts with favorable immunological properties, develop schedules of Flt3-L administration that increase the frequency and potency of immune-regulatory DC, and use bio- luminescent and fluorescent imaging to track in vivo homing, expansion, and function of donor DC in mouse BMT models. This research will yield a mechanistic understanding of how donor DC subset(s) regulate immunity after allogeneic stem cell transplant that will inform studies in solid organ transplantation and adoptive T cell therapies such as CART. Positive results can lead to a clinical trial of Flt3-L treatment of stem cell donors.